脂肪酶高产菌的选育、酶的纯化和表征以及两种诱导方式产酶的机理研究

发布时间：2018-09-01 17:40

【摘要】：脂肪酶是一种既可催化水解反应又可催化合成反应的生物催化剂。因微生物脂肪酶的产量高,便于基因操作,生产无季节波动等原因,比植物和动物来源的脂肪酶的应用更为广泛。本研究依靠罗丹明B橄榄油初筛培养基从5份土样中筛选到16株细菌和16株真菌。通过测定发酵液酶活和专一性,对菌株进行复筛,并利用SHERLOCK(?)全自动微生物鉴定系统、26-28S rDNA或ITS序列测定等方法进行鉴定。复筛出7株产酶较优菌,其中Pseudomonas sp. B1-1、 Acinetobacter sp. B1-2、Acinetobacter sp. B5-1、Trichosporon sp. F1-2为sn-1(3)位专一性,Staphylococcus sp. B2-1、Acinetobacter sp. B3-8和Galactomyces candidum F1-1无专一性。所有分离菌中T. sp. F1-2的酶活力最高,因此选为重点研究对象。系统发育分析显示其与T. cacaoliposimilis和T. laibachii的进化关系最近。研究发现,T. sp. F1-2的胞外比酶活高于胞内,所以更有利于纯化。通过50%饱和度的硫酸铵沉淀、8000-14000 Da透析和DEAE-sepharose FF弱阴离子交换柱层析(pH 8.3)的纯化步骤,将T. sp F1-2脂肪酶纯化了3.96倍,比酶活达到223.13 U/mmg,经过SDS-PAGE分析,测得其分子量为32.6 kDa。本论文详细研究了T. spF1-2脂肪酶的性质。该酶能长期耐受的最高温度为45℃,短期最适反应温度为50℃,其最适保藏pH范围为7.0～9.0,最适反应pH为8.0。该酶具有sn-1(3)位专一性。其对底物的链长也具有明显的选择性,最优的底物是辛酸酯；该酶在Na+、K+、Ca2+、Mg2+和M112+等金属离子中酶活稳定性较好,Zn2+是其最强的酶活抑制剂。该酶对各种类型的表面活性剂都很敏感,但对非离子型表面活性剂的耐受性稍好于阴离子表面活性剂。该酶对多种有机溶剂都表现出非常突出的耐受性,乙醚、二氯甲烷、甲苯和正己烷还能一定程度上提升其酶活,其对强极性试剂丙三醇、二甲基亚砜和甲醇的高耐受性在脂肪酶中较少见。深入研究了两种诱导方式,即外加诱导油和自身合成油脂分别为产酶诱导物。当添加诱导油发酵时,菌体会转运诱导油到胞内,形成球状的脂质体,并且在转运前先将诱导油乳化成极小的油滴,以便于转运。低浓度葡萄糖发酵时,由于第一碳源很快耗尽,转运在发酵24 h时已经很显著。高浓度葡萄糖发酵时,虽然并不缺乏葡萄糖,也会在稍晚于低浓度发酵一段时间后开始快速转运油脂,形成大量脂质体,这很可能便是高糖发酵时产酶仍维持高水平的原因。经过脂肪酸组成分析确认,诱导油发酵时,胞内脂质体的脂肪酸组成与诱导油一致,确系来自胞外转运,这与葡萄糖的初始浓度无关。当不添加诱导油发酵时,该菌仍会通过转化葡萄糖形成较多的脂质体。由于合成脂类需要脂肪酶参与,且合成后积累在体内又成了自产的诱导油,进一步促进脂肪酶的生成,因此,即使不添加诱导油,该菌也能生产一定量的脂肪酶。该菌自产的脂类主要含五种脂肪酸：肉豆蔻酸、棕榈酸、硬脂酸、油酸和亚油酸。降低发酵培养基中的氮源浓度,有利于油脂积累,且会影响脂肪酸的组成,使得饱和脂肪酸的含量增加,不饱和脂肪酸的含量减少。对比两种诱导途径,直接添加诱导油发酵产酶的效率要高于自身产油诱导的效率,且采用前种发酵时,从种子培养就开始添加诱导油会显著提高产酶量,而对于后种发酵,种子培养基中添加油并不会提高胞外产酶。生产能力偏低是限制T. sp F1-2实现工业化生产的主要原因。于是利用常压室温等离子体对野生菌进行了诱变处理,并建立了96孔板培养结合对硝基苯酚棕榈酸酯法测定酶活力的高通量筛选方法,实现了60个突变菌株的初筛。以酶活力为筛选指标时,突变率和正突变率分别为51.7%和28.3%。8株初筛菌株的摇瓶发酵结果显示,A13和A5的产酶提高最显著,培养96h后分别比野生菌增加2.64倍和1.54倍,且两个突变菌株的遗传稳定性良好。进一步的对比研究发现,突变菌株A13相较野生菌的最大优势在于提前24 h便能达到最高产酶量。本论文还利用商业化酶制剂研究了脂肪酶的两种重要特性,位置专一性和酰基迁移。脂肪酶的位置专一性在结构酯的合成和油脂改性中具有重要意义。由于水体系和非水体系的差异,传统的水解判定法得出的专一性与该酶在合成反应中的表现可能并不一致。于是建立了利用月桂酸和山茶油的酸解反应来直接评估酶在无溶剂体系中位置专一性的方法。利用此方法,Lipozyme RM IM、L02、L03和L04被鉴定为sn-1(3)位专一性,L01为弱专一性,Novozym 435近似无专一性。通过替换酶的底物,模型反应的可预测性得到验证。根据酸解法和水解法结果的对比分析,两种条件下酶的位置专一性通常是相同的,除了易受到溶剂体系影响的Novozym 435。因此,新方法能够避免水解判定结果在合成反应中应用的局限性。除了酸解反应模型,还建立了一个酯交换反应模型来研究酰基迁移的影响因素。模型反应的底物为等摩尔量的三月桂酸甘油酯和1,3-棕榈酸-2-油酸甘油酯,三种固定化脂肪酶参与了此反应。通过测定甘油三酯的组成和脂肪酸的分布来检测sn-1(3)位的酯交换和sn-2位的酰基迁移。固定化于聚丙烯的Rhizopus oryzae脂肪酶表现出非常严格的sn-1(3)位专一性,2位发生的改变非常小。而固定化于二氧化硅的Thermomyces lanuginosus脂肪酶(Lipozyme(?) TLIM)能在24 h内完成完全的随机化。固定化于聚丙烯的T. lanuginosus脂肪酶能催化2位发生中等程度的改变。因此,T. lanuginosus脂肪酶和二氧化硅会促进脂肪酸分布的随机化,而R. oryzae脂肪酶和聚丙烯则不会。高水分活度促进水解因此会增加不完整甘油酯的浓度,但同时也会抑制这些中间产物的的酰基迁移,最后的结果是,当酯交换率的程度相同时,不同水分活度下2位的酰基迁移没有显著差异,而低水分活度的主要优势是能保证甘油三酯的产量。
[Abstract]:Lipase is a kind of biocatalyst which can catalyze both hydrolysis and synthesis reactions. Microbial lipase is more widely used than plant and animal lipase because of its high yield, easy gene manipulation and no seasonal fluctuation in production. This study relied on Rhodamine B olive oil primary screening medium to screen five soil samples. Sixteen strains of bacteria and 16 fungi were screened by determining enzyme activity and specificity of fermentation broth, and identified by SHERLOCK (?) automatic microbial identification system, 26-28S rDNA or ITS sequencing. Seven strains of bacteria with better enzyme production were screened out, including Pseudomonas sp.B1-1, Acinetobacter sp.B1-2, Acinetobacter sp.B5-1, Tri. Chosporon sp.F1-2 is sn-1(3) site specific, Staphylococcus sp.B2-1, Acinetobacter sp.B3-8 and Galactomyces candidum F1-1 are not. T.sp.F1-2 has the highest enzyme activity in all isolates, so it is selected as the key research object. Phylogenetic analysis shows that it has the closest evolutionary relationship with T.caoliposimilis and T.baclaihii. T.sp.F1-2 lipase was purified by 50% ammonium sulfate precipitation, 8000-14000 Da dialysis and DEAE-sepharose FF weak anion exchange column chromatography (pH 8.3). The specific activity of T.sp.F1-2 lipase was 3.96 times and 223.13 U/mmg, respectively. The molecular weight of T.spF1-2 lipase was 32.6 kDa.The properties of T.spF1-2 lipase were studied in detail in this paper.The optimum temperature for long-term tolerance of T.spF1-2 lipase was 45, the optimum reaction temperature was 50, the optimum preservation pH ranged from 7.0 to 9.0, and the optimum reaction pH was 8.0. Zinc 2+ is the strongest inhibitor of the enzyme activity. The enzyme is sensitive to various types of surfactants, but has a slightly better tolerance to nonionic surfactants than anionic surfactants. The enzyme exhibits good stability in various organic solvents. Ethyl ether, dichloromethane, toluene and n-hexane can also enhance their enzyme activity to a certain extent, and their high tolerance to strong polar reagents glycerol, dimethyl sulfoxide and methanol is rare in lipase. Two induction methods, i.e. external induction oil and self-synthesized oil, are studied in depth, which are enzyme-producing inducers respectively. In low concentration glucose fermentation, the first carbon source is quickly exhausted, and the translocation is already significant at 24 h of fermentation. Lack of glucose also causes the rapid translocation of lipids and the formation of a large number of liposomes after a period of fermentation at a later time than at a lower concentration, which may be the reason why enzyme production remains high during high glucose fermentation. This is not related to the initial concentration of glucose. When induced oil is not added, the bacteria can still form more liposomes by converting glucose. Because lipase is involved in the synthesis of lipids and accumulated in the body after synthesis, it becomes a self-produced inducing oil, which further promotes the production of lipase, even without the addition of inducing oil. The lipids produced by the bacteria mainly contain five kinds of fatty acids: myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid. Reducing the concentration of nitrogen source in the fermentation medium is beneficial to the accumulation of lipids, and will affect the composition of fatty acids, which will increase the content of saturated fatty acids and decrease the content of unsaturated fatty acids. Compared with the two induction pathways, the efficiency of enzyme production by direct addition of induction oil was higher than that by self-induction. Adding induction oil from the beginning of seed culture could significantly increase the enzyme production by using pre-seed fermentation, but for post-seed fermentation, adding oil to seed medium would not increase the extracellular enzyme production. The main reason for industrialized production of T.sp F1-2 was that the wild bacteria were mutagenized by atmospheric pressure room temperature plasma and a high throughput screening method was established by 96-well plate culture combined with p-nitrophenol palmitate method for the determination of enzyme activity. The results of shaking flask fermentation showed that the enzyme production of A13 and A5 increased by 2.64 times and 1.54 times respectively after 96 h culture, and the genetic stability of the two mutant strains was good. Two important properties of lipase, location specificity and acyl migration, were studied by commercial enzyme preparations. The location specificity of lipase is very important in the synthesis of structural esters and the modification of lipids. The specificity of Lipozyme RM IM, L02, L03 and L04 was identified as sn-1(3) site specificity, L01 as weak specificity, Novozym 435 as position specificity by acid hydrolysis of lauric acid and Camellia oil. The predictability of the model reaction was validated by substituting the substrates of the enzymes. According to the comparative analysis of the results of acid hydrolysis and hydrolysis, the site specificity of the enzymes under the two conditions is usually the same, except for Novozym 435, which is susceptible to the influence of the solvent system. Limitations of application. In addition to the acidolysis model, a transesterification model was established to study the factors affecting the migration of acyl groups. The substrate of the model reaction was equal molar amounts of glycerol laurate and 1,3-palmitic acid-2-oleic acid glycerol ester. Three immobilized lipases participated in the reaction. The composition of triglycerides was determined. Fatty acid distribution was used to detect the transesterification of sn-1(3) and the migration of acyl groups at sn-2. The immobilized polypropylene-based Rhizopus oryzae lipase showed very strict sn-1(3) site specificity, with very small changes in the two sites. The immobilized silica-based Thermomyces lanuginosus lipase (Lipozyme (?) TLIM) could be completed within 24 hours. Complete randomization. T. lanuginosus lipase immobilized on polypropylene catalyzes moderate changes in the two sites. Therefore, T. lanuginosus lipase and silica promote randomization of fatty acid distribution, while R. oryzae lipase and polypropylene do not. High water activity promotes hydrolysis and therefore increases the concentration of incomplete glycerides. But it also inhibits the acyl migration of these intermediates. The final result is that when the degree of transesterification is the same, there is no significant difference between the two acyl migration under different water activity, and the main advantage of low water activity is to ensure the yield of triglycerides.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ925;Q814

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